Design, Synthesis, and Anti-Hepatocellular Carcinoma Evaluation of Sesquiterpene Lactone Epimers Trilobolide-6-O-isobutyrate Analogs

Hepatocellular carcinoma (HCC), one of the most common malignant cancers with a low 5-year survival rate, is the third leading cause of cancer-related deaths worldwide. The finding of novel agents and strategies for the treatment of HCC is an urgent need. Sesquiterpene lactones (SLs) have attracted extensive attention because of their potent antitumor activity. In this study, a new series of SL derivatives (3–18) were synthesized using epimers 1 and 2 as parent molecules, isolated from Sphagneticola trilobata, and evaluated for their anti-HCC activity. Furthermore, the structures of 4, 6, and 14 were confirmed by X-ray single-crystal diffraction analyses. The cytotoxic activities of 3–18 on two HCC cell lines, including HepG2 and Huh7, were evaluated using the CCK-8 assay. Among them, compound 10 exhibited the best activity against the HepG2 and Huh7 cell lines. Further studies showed that 10 induced cell apoptosis, arrested the cell cycle at the S phase, and induced the inhibition of cell proliferation and migration in HepG2 and Huh7. In addition, absorption, distribution, metabolism, and excretion (ADME) properties prediction showed that 10 may possess the properties to be a drug candidate. Thus, 10 may be a promising lead compound for the treatment of HCC.


Introduction
Cancer is a persistent unmet global challenge claiming millions of lives yearly and taking a tremendous toll on healthcare services and world economies [1].According to the 2020 WHO report, liver cancer has the sixth highest incidence rate (4.7%) and the third highest mortality rate (8.3%) among the 36 types of cancer, making it one of the deadliest forms of cancer [2].The most common primary liver cancer, hepatocellular carcinoma (HCC), is one of the most prevalent and lethal tumors [3].As our understanding of HCC biology forges ahead, a number of first-and second-line chemotherapeutic drugs, as well as new targeted agents, immune checkpoint inhibitors (ICIs), and their combination therapies, are among the options available to patients [4].Despite progress in this field, the effectiveness of most of them is severely limited by drug resistance, as well as invasion and metastasis of cancers [5,6].The development of new effective anti-HCC drugs is therefore essential.
Natural sesquiterpene lactones (SLs) are a family of secondary metabolites derived almost exclusively from Asteraceae/Compositae plants [7].These natural products have been a hot topic in the field of tumor drug research because of their complexity, diversity, novelty of structures, and uniqueness of function [8].In recent years, SL-derived drugs such as derivatives of Artemisinin (ART) and Parthenolide (PTL) (Figure 1), have shown promise for the potential treatment of various types of cancer [9][10][11][12][13][14]. ART and its derivatives such as dihydroartemisinin (DHA) and artesunate could promote cancer cell apoptosis, induce cell cycle arrest and autophagy, and inhibit cancer cell invasion as well as migration, and they represent promising candidates for cancer therapy, supported by some clinical phase I/II trials [15][16][17][18].PTL showed promising anti-cancer properties.Nevertheless, PTL has some disadvantages, such as poor oral bioavailability and poor water solubility [19,20].DMAPT, a derivative of PTL, effectively increased the water solubility and oral bioavailability, and has advanced into a phase I clinical trial for the treatment of acute myeloid leukemia [21].And the other PTL derivative, ACT001, has been clinically tested in Australia and China for the treatment of glioblastoma [22].
Molecules 2024, 29, x FOR PEER REVIEW 2 of 20 Natural sesquiterpene lactones (SLs) are a family of secondary metabolites derived almost exclusively from Asteraceae/Compositae plants [7].These natural products have been a hot topic in the field of tumor drug research because of their complexity, diversity, novelty of structures, and uniqueness of function [8].In recent years, SL-derived drugs such as derivatives of Artemisinin (ART) and Parthenolide (PTL) (Figure 1), have shown promise for the potential treatment of various types of cancer [9][10][11][12][13][14]. ART and its derivatives such as dihydroartemisinin (DHA) and artesunate could promote cancer cell apoptosis, induce cell cycle arrest and autophagy, and inhibit cancer cell invasion as well as migration, and they represent promising candidates for cancer therapy, supported by some clinical phase I/II trials [15][16][17][18].PTL showed promising anti-cancer properties.Nevertheless, PTL has some disadvantages, such as poor oral bioavailability and poor water solubility [19,20].DMAPT, a derivative of PTL, effectively increased the water solubility and oral bioavailability, and has advanced into a phase I clinical trial for the treatment of acute myeloid leukemia [21].And the other PTL derivative, ACT001, has been clinically tested in Australia and China for the treatment of glioblastoma [22].SLs' cytotoxic activity has been described as mainly dependent on the α-methyleneγ-lactone group, which is prone to react with suitable nucleophiles [23][24][25].Trilobolide-6-O-isobutyrate A and B (1 and 2), a pair of C-5 epimers of SLs with high oxygen content, were isolated from the flowers of S. trilobata, they also have an α-methylene-γ-lactone group .Subsequent investigations indicated that 2 could strongly inhibit the proliferation of HCC through inhibition of the IL-6/STAT3 signaling pathway [26].And a new series of SL derivatives were designed and synthesized using 1 and 2 as the parent molecules.Unfortunately, all of the compounds did not show significant in vitro antiproliferative activity against the tested human cancer cell lines [27].
Based on our ongoing pursuit of the investigation of 1 and 2 to further enhance their antitumor profile, a series of 1 and 2 derivatives (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) were further designed and synthesized in this work.All the compounds were evaluated for their potential inhibition of HCC cell lines.We further investigated the effects of the most active compound 10 on HepG2 and Huh7 cells, and computational absorption, distribution, metabolism, and excretion (ADME) estimation studies were performed.SLs' cytotoxic activity has been described as mainly dependent on the α-methyleneγ-lactone group, which is prone to react with suitable nucleophiles [23][24][25].Trilobolide-6-O-isobutyrate A and B (1 and 2), a pair of C-5 epimers of SLs with high oxygen content, were isolated from the flowers of S. trilobata, they also have an α-methylene-γ-lactone group.Subsequent investigations indicated that 2 could strongly inhibit the proliferation of HCC through inhibition of the IL-6/STAT3 signaling pathway [26].And a new series of SL derivatives were designed and synthesized using 1 and 2 as the parent molecules.Unfortunately, all of the compounds did not show significant in vitro antiproliferative activity against the tested human cancer cell lines [27].
Based on our ongoing pursuit of the investigation of 1 and 2 to further enhance their antitumor profile, a series of 1 and 2 derivatives (3-18) were further designed and synthesized in this work.All the compounds were evaluated for their potential inhibition of HCC cell lines.We further investigated the effects of the most active compound 10 on HepG2 and Huh7 cells, and computational absorption, distribution, metabolism, and excretion (ADME) estimation studies were performed.

Synthesis
Compounds 1 and 2 were isolated from the EtOH solutions of the dried flowers of S. trilobata by repeated column chromatography (CC) and these two compounds were further purified by recrystallization from EtOH following previously described methods [28].In order to investigate whether the presence of ester groups and terminal double bonds in 1 and 2 has a positive effect on anti-hepatocellular carcinoma activities, we designed synthetic routes primarily considering the hydrolysis reaction.The acetyl groups at the C-1 and C-9 positions and the isobutyryl group at the C-6 position were removed, and two acetyl groups and one isobutyryl substituent on 1 and 2 were removed by selecting different acid and base systems to obtain hydrolysates with different degrees of ester removal.We used NaBH 4 as a reductant to reduce the exocyclic double bond of C-11 (13) to the methyl group.Schemes 1 and 2 represent the synthetic pathways of all the compounds, respectively.

Synthesis
Compounds 1 and 2 were isolated from the EtOH solutions of the dried flowers of S. trilobata by repeated column chromatography (CC) and these two compounds were further purified by recrystallization from EtOH following previously described methods [28].In order to investigate whether the presence of ester groups and terminal double bonds in 1 and 2 has a positive effect on anti-hepatocellular carcinoma activities, we designed synthetic routes primarily considering the hydrolysis reaction.The acetyl groups at the C-1 and C-9 positions and the isobutyryl group at the C-6 position were removed, and two acetyl groups and one isobutyryl substituent on 1 and 2 were removed by selecting different acid and base systems to obtain hydrolysates with different degrees of ester removal.We used NaBH4 as a reductant to reduce the exocyclic double bond of C-11 (13) to the methyl group.Schemes 1 and 2 represent the synthetic pathways of all the compounds, respectively.First, 1 was dissolved in THF and hydrolyzed by HCl solution.The crude product was further purified by silica gel CC to obtain pure products 3 and 4 [29].Then, 1 was dissolved in THF and H 2 O, the hydrolysis condition was changed to alkaline KOH solution, and the reaction was at 0 • C for 2 h to prepare the pure product 5. Subsequently, the appropriate 1 and NaBH 4 solids were dissolved in the CH 3 CN and stirred at 60 • C for 26.5 h to afford pure products 6 and 7 by HPLC [30].In order to obtain different degrees of removal of the ester groups at the C-1, C-6, and C-9 positions and expose the hydroxyl groups, we switched to using KOH to provide alkaline conditions and MeOH as the solvent.The reaction mixture was stirred at room temperature for 6 h.Then, crude products were further purified by silica gel CC to afford pure products 8 and 9.In the hydrolysis of 2, we also optimized different acid-base systems under the conditions of HCl or H 2 SO 4 solution, and prepared the products 10 and 12.The hydrolysis derivatives 13-16 of 2 were also prepared according to the synthesis method of compounds 6-9.Finally, 2 was dissolved in DMSO and reacted in the presence of DBU to obtain products 17 and 18 [31].First, 1 was dissolved in THF and hydrolyzed by HCl solution.The crude product was further purified by silica gel CC to obtain pure products 3 and 4 [29].Then, 1 was dissolved in THF and H2O, the hydrolysis condition was changed to alkaline KOH solution, and the reaction was at 0 °C for 2 h to prepare the pure product 5. Subsequently, the appropriate 1 and NaBH4 solids were dissolved in the CH3CN and stirred at 60 °C for 26.5 h to afford pure products 6 and 7 by HPLC [30].In order to obtain different degrees of removal of the ester groups at the C-1, C-6, and C-9 positions and expose the hydroxyl groups, we switched to using KOH to provide alkaline conditions and MeOH as the solvent.The reaction mixture was stirred at room temperature for 6 h.Then, crude products were further purified by silica gel CC to afford pure products 8 and 9.In the hydrolysis of 2, we also optimized different acid-base systems under the conditions of HCl or H2SO4 solution, and prepared the products 10 and 12.The hydrolysis derivatives 13-16 of 2 were also prepared according to the synthesis method of compounds 6-9.Finally, 2 was dissolved in DMSO and reacted in the presence of DBU to obtain products 17 and 18 [31].
The structures of these derivatives were firmly established by combination with the physical and chemical properties, and 1D/2D NMR and HRMS methods.The Michael addition at the α-methylene-γ-lactone of SLs with amines has been found to be highly stereospecific and yielded exclusively a single stereoisomer with the (R*)-configuration at the newly formed C-11 chiral carbon.This was attributed to the stereo-exclusive protonation The structures of these derivatives were firmly established by combination with the physical and chemical properties, and 1D/2D NMR and HRMS methods.The Michael addition at the α-methylene-γ-lactone of SLs with amines has been found to be highly stereo-specific and yielded exclusively a single stereoisomer with the (R*)-configuration at the newly formed C-11 chiral carbon.This was attributed to the stereo-exclusive protonation of the enolate formed during the Michael addition [32].In this research, firstly, the absolute configuration of the parent molecules 1 and 2 was determined by X-ray crystal analysis [26].Secondly, in our previous work, the 11R stereochemistry of the SLs derivatives was confirmed by the NOE effect between H-11 and H-6 [27].Finally, the X-ray crystal structures of 4, 6, and 14 (Figure S55) further confirmed the R configuration at C-11.

Biological Evaluation 2.2.1. Cell Viability Assay and Structure-Activity Relationships (SARs)
The anti-HCC activity of 1, 2, and their derivatives was evaluated by the CCK-8 assay against human HCC cell lines HepG2 and Huh7, and compared with adriamycin as the working standard.As shown in Table 1, some derivatives showed comparatively significant IC 50 values compared to 1 and 2. On the basis of the above activity data, a preliminary SARs was deduced.In the hydrolysates of 1 and 2, the cytotoxicity of compounds 3 and 10 was comparable or better than that of the parent molecules.It was suggested that the double bonds at the C-4, 14 or C-3, 4 positions were more favorable to enhancing the activity of HCC.Notably, the double bond between C-11 and C-13 was reduced to the methyl group in all the compounds, which were less potent than the parental compounds, further confirming SLs' cytotoxic activity as being mainly dependent on the α-methylene-γ-lactone group [23][24][25].Compound 10 demonstrated the best antiproliferative activity against HepG2 and Huh7 cells with IC 50 values of 9.73 and 18.86 µM, respectively.Therefore, 10 was selected for subsequent antitumor experiments on HepG2 and Huh7 cell lines in vitro.

Anti-Proliferative Effect of Compound 10 on Huh7 and HepG2 Cells
The effects of different concentrations of compound 10 on two types of human HCC cell lines (Huh7 and HepG2) were detected by CCK-8 assay.The results indicated that 10 could dose-dependently decrease the viability of the two HCC cell lines (Figure 2A,B).In addition, 10 could also inhibit the viability of Huh7 cells in time-and dose-dependent manners (Figure 2C,D).The live/dead cell staining assay (Calcein-AM/PI double staining kit) was also employed to evaluate the in vitro cytotoxicity of 10.In the control, a bright green fluorescence (calcein AM for live cells probe) and a few red dots with fluorescence (PI for dead cells probe) were found in Huh7 and HepG2 cells (Figure 3).A decreased number of live cells for both Huh7 and HepG2 was obviously found in 10, whereas that of dead cells increased, compared with the control.Thus, the fluorescence imaging results were in agreement with those of the cytotoxicity assay.
The colony-forming assay is one of the effective methods to measure cell proliferation.When a single cell lasts for more than six generations in vitro, the descendants of the cell population are called clones.At this time, each clone contains more than 50 cells, and the size is between 0.3 and 1.0 mm.By counting the clone formation rate, the proliferation potential of a single cell can be quantitatively analyzed, and the proliferation ability and independent survival ability of the cell can be understood.Consequently, this work performed a clone-forming assay to investigate whether compound 10 inhibited proliferation.Huh7 and HepG2 cells were first incubated using 10 for a 24 h period, respectively, and followed by 14 d culture to observe a visible colony.According to Figure 4, Huh7 and HepG2 cells had significantly suppressed clone-forming capacity after 10 exposure, which was consistent with the CCK-8 assays.independent survival ability of the cell can be understood.Consequently, this work performed a clone-forming assay to investigate whether compound 10 inhibited proliferation.Huh7 and HepG2 cells were first incubated using 10 for a 24 h period, respectively, and followed by 14 d culture to observe a visible colony.According to Figure 4, Huh7 and HepG2 cells had significantly suppressed clone-forming capacity after 10 exposure, which was consistent with the CCK-8 assays.

Compound 10 Promoted Cell Apoptosis of Huh7 and HepG2 Cells
In order to investigate the proapoptotic effect of compound 10, Huh7 and HepG2 cells were stained with Annexin V-FITC and PI probe, and subjected to flow cytometry analysis.Both cells were incubated with different concentrations of 10 for 24 h.In comparison to the control group, all treatment groups of 10 significantly induced apoptosis of Huh7 and HepG2 cells (Figure 5).The percentages of total apoptotic cells increased significantly from 6.3% (control, Huh7 cells) and 6.51% (control, HepG2 cells) to 67.8% (40 µM, Huh7 cells) and 24% (10 µM, HepG2 cells), respectively.Compound 10 induced approximately 30% late apoptosis of Huh7 cells at concentrations of 40 µM, and late apoptosis was more obvious.The results established that compound 10 could significantly induce Huh7 and HepG2 cell apoptosis.

Compound 10 Promoted Cell Apoptosis of Huh7 and HepG2 Cells
In order to investigate the proapoptotic effect of compound 10, Huh7 and HepG2 cells were stained with Annexin V-FITC and PI probe, and subjected to flow cytometry analysis.Both cells were incubated with different concentrations of 10 for 24 h.In comparison to the control group, all treatment groups of 10 significantly induced apoptosis of Huh7 and HepG2 cells (Figure 5).The percentages of total apoptotic cells increased significantly from 6.3% (control, Huh7 cells) and 6.51% (control, HepG2 cells) to 67.8% (40 µM, Huh7 cells) and 24% (10 µM, HepG2 cells), respectively.Compound 10 induced approximately 30% late apoptosis of Huh7 cells at concentrations of 40 µM, and late apoptosis was more obvious.The results established that compound 10 could significantly induce Huh7 and HepG2 cell apoptosis.

Compound 10 Arrested the Cell Cycle in the S Phase in Huh7 and HepG2 Cells
The cell cycle plays an important role in the operation and development of cell life due to its regulation of the division and duplication of DNA.Therefore, blocking the cell cycle is considered an effective way to eliminate tumor cells.To demonstrate the antiproliferative effect of compound 10 on the cell cycle process, flow cytometry was used to monitor PI staining treatment with 10 for 24 h; this led to an increased percentage of cells in the S phase from 39.76% and 33.22% of the untreated control to 46.27% and 36.91%,respectively (Figure 6A,B and 6C,D).This result demonstrated that 10 arrested Huh7 and HepG2 cell cycle progression in the S phase.The cell cycle plays an important role in the operation and development of cell life due to its regulation of the division and duplication of DNA.Therefore, blocking the cell cycle is considered an effective way to eliminate tumor cells.To demonstrate the antiproliferative effect of compound 10 on the cell cycle process, flow cytometry was used to monitor PI staining treatment with 10 for 24 h; this led to an increased percentage of cells in the S phase from 39.76% and 33.22% of the untreated control to 46.27% and 36.91%,respectively (Figure 6A,B and 6C,D).This result demonstrated that 10 arrested Huh7 and HepG2 cell cycle progression in the S phase.

Effect of Compound 10 on Cell Migration
Migration is one of the essential links in the process of tumor cell metastasis.For tumor cells to separate from the mother tumor, cross the blood vessel wall, and invade the surrounding normal tissue, they need some exercise ability.Tumor cells that are highly metastatic usually have high motility.The cell scratch method and transwell migration are simple methods to determine the migration, movement, and repair ability of cells.Thus, the effects of 10 on cell migration were further investigated.As shown in Figure 7A,B, 10 significantly inhibited the rate of wound healing in Huh7 cells after treatment at the indicated concentrations for 48 h.The number of cells that went through the membrane in transwell assays also decreased in a dose-dependent manner (Figure 7C,D).The above results suggested that 10 could effectively suppress the migration of Huh7 cells in a dosedependent manner.

ADME Prediction
Many drug molecule candidates remain in phase studies without a drug molecule due to poor ADME properties.Performing theoretical ADME calculations for designed and newly synthesized compounds may aid the progression of these compounds in advanced in vitro and in vivo studies [33,34].Therefore, some properties of the designed compounds, such as physicochemical, lipophilicity, water-solubility, pharmacokinetics, drug-likeness, and medicinal chemistry properties, were calculated using SwissADME online tools, and some of the most active properties of 10 are given in Table 2.The druglikeness assessment of 10 was performed by predicting Lipinski's rule of five, which includes molecular weights (MW < 500), lipophilicity (log Po/w < 5), number of hydrogen bond acceptors (HBA ≤ 10), and number of hydrogen bond donors (HBD ≤ 5) to determine

ADME Prediction
Many drug molecule candidates remain in phase studies without a drug molecule due to poor ADME properties.Performing theoretical ADME calculations for designed and newly synthesized compounds may aid the progression of these compounds in advanced in vitro and in vivo studies [33,34].Therefore, some properties of the designed compounds, such as physicochemical, lipophilicity, water-solubility, pharmacokinetics, drug-likeness, and medicinal chemistry properties, were calculated using SwissADME online tools, and some of the most active properties of 10 are given in Table 2.The drug-likeness assessment of 10 was performed by predicting Lipinski's rule of five, which includes molecular weights (MW < 500), lipophilicity (log Po/w < 5), number of hydrogen bond acceptors (HBA ≤ 10), and number of hydrogen bond donors (HBD ≤ 5) to determine the "drug-likeness" of 10.Its solubility in water is fine and favorable.Its gastrointestinal absorption was calculated as high and not able to pass through the blood-brain barrier.The findings of ADME showed that 10 has very good drug-like properties.

General Experimental Procedures
The 1D and 2D NMR experiments were performed on a Bruker AV-400 (Bruker Corporation, Zurich, Switzerland) instrument with tetramethylsilane as the internal standard.HR-ESI-MS spectra were acquired on a Bruker Daltonics Apex-Ultra 7.0 T (Bruker Corporation, Billerica, MA, USA) and a Q-TOF Ultima Global GAA076 LC mass spectrometer.Single-crystal data were measured with an Agilent Gemini Ultra X-ray single-crystal diffractometer (Cu Kα radiation).Preparative HPLC was conducted using an Agilent 1260 prep-HPLC system with a Waters C18 analytical HPLC column (4.6 × 250 mm, 5 µm) and a semipreparative column (9.4 × 250 mm, 7 µm).Sephadex LH-20 (Pharmacia Co. Ltd., Sandwich, UK) and silica gel (200-300 and 300-400 mesh, Qingdao Marine Chemical Inc., Qingdao, China) were used for column chromatography (CC).All solvents were purchased from Xilong Chemical Reagent Factory (Guangzhou, China).The flowers of Sphagneticola trilobata were collected from Haikou County, Hainan Province, China, in August 2018, and identified by Professor Qiong-Xin Zhong, School of Life Science, Hainan Normal University.

The Separation of SLs 1 and 2
The isolation process of 1 and 2 is shown in the supporting information.

General Procedure for the Synthesis of 1 Hydrolysis Derivatives 3-4
In a 25 mL round-bottomed flask, 712 mg (1.157 mmol) of 1, 2 mL of THF, and 2 mL of dilute HCl (2 mol/L) were added sequentially.The reaction was heated under reflux at 45 • C for 26 h.After the reaction was complete (CHCl 3 :acetone = 6:1), the reaction was detected by TLC.The pH was adjusted to 6-7 with potassium carbonate solution, and products 3 and 4 were separated by CC (CHCl 3 :acetone = 8:1).

General Procedure for the Synthesis of 1 Hydrolysis Derivative 5
Add 125.0 mg (0.277 mmol) 1 to a 50 mL round bottom flask and dissolve in 6 mL THF and 10 mL H 2 O. Add 4 mL KOH solution (0.5 mol/L) slowly at 0 • C, stirring for 2 h.After the reaction was completely detected by TLC (Petroleum ether:acetone = 1.5:1), the mixture was adjusted to pH 2-3 with 10% dilute HCl, and product 5 was purified by CC (petroleum ether:acetone = 3:1).

General Procedure for the Synthesis of 1 Hydrolysis Derivatives 6-9
An amount of 297 mg (0.657 mmol) of compound 1 and 88 mg of NaBH4 were dissolved in 50 mL of CH 3 CN and stirred at 60 • C for 26.5 h.After the reaction was complete (CHCl 3 : acetone = 6: 1) as detected by TLC, the pH of the mixture was adjusted to 6-7 with 10% dilute HCl, extracted with EtOAc for three times, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and prepared by HPLC to obtain compounds 6 and 7. Subsequently, 23.3 mg (0.051 mmol) of 6 and 22.6 mg KOH were dissolved in 5 mL of MeOH, stirred at room temperature for 6 h, and the reaction was detected by TLC (CHCl 3 :acetone = 3:1).The products 8 and 9 were purified by CC (CHCl 3 :acetone = 6:1).

General Procedure for the Synthesis of 2 Hydrolysis Derivatives 10-12
Referring to the synthesis method of 3 and 4, compounds 10 and 11 were obtained by CC purification (CHCl 3 :acetone = 3:1).For further optimization of acid conditions, we replaced the previous acid with a dilute H 2 SO 4 solution (8 mol/L) and refluxed the mixture at 45 • C for 10 h.After the reaction was completed (CHCl 3 :acetone = 6:1), the products 10, 11, and 12 were separated by CC (CHCl 3 :acetone = 8:1).

General Procedure for the Synthesis of 2 Hydrolysis Derivatives 13-16
According to the synthesis of 6 and 7, compounds 13 and 14 were prepared by HPLC.According to the synthesis of 8 and 9, compounds 15 and 16 were purified by CC (CHCl 3 :acetone = 3:1).

General Procedure for the Synthesis of 2 Hydrolysis Derivatives 17-18
The appropriate amounts of 2 (0.177 mmol) and DBU (100 ul) were dissolved in DMSO (2 mL) for the reaction.The reaction mixture was stirred at room temperature for 79 h.The pH of the mixture was adjusted to 6-7 with 10% dilute hydrochloric acid and the products 17 and 18 were purified by HPLC.

Cell Apoptosis Analysis
Huh7 and HepG2 cells were seeded in 6-well cell culture plates.The cells were then grown for 24 h with varied doses of 10, and subsequent processes were carried out based on the explanatory memorandum of the Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen, Cat.#: 40302ES60, Shanghai, China).Briefly, cells were digested by trypsin, centrifuged at 4 • C with 1500 rpm for 5 min, washed with pre-cooling PBS twice, and resuspended in 100 µL 1 × binding buffer before staining with 5 µL Annexin-V-FITC and 10 µL PI staining solution for 10-15 min incubation in the darkness.After diluting with 400 µL of 1 × binding buffer, the samples were examined by flow cytometry (Sysmex-Partec CyFlow TM Cube 6).Data were analyzed using FlowJo V1 (Flexera Software, Chicago, IL, USA).

Cell Cycle Arrest Analysis
Huh7 and HepG2 cells were seeded in 6-well cell culture plates.The cells were then exposed to various doses of 10 for 24 h.The cells were harvested, washed three times with PBS, and then fixed for at least 12 h in cold 70% ethanol at 4 • C.After that, all of the cells were washed three times in PBS to remove any remaining ethanol.The cells were then resuspended in the solution stained with propidium iodide and incubated at room temperature for 30 min in the dark.A NovoCyte Flow Cytometer (Agilent, Santa Clara, CA, USA) was used to analyze the samples.

Wound Healing Assay
Cells (1 × 10 6 /mL) were seeded in 6-well plates and cultured until each well was covered.The cell monolayer was scraped vertically with a 200 µL sterile pipette tip and rinsed 3 times with sterile PBS.After that, 5% FBS medium was added with the compounds to be tested and incubated for 48 h.Images were taken at 0 and 48 h for each scratch by microscope (OLYMPUS IX-51).ImageJ software was used to compare the edge-by-edge measurements at 0 h and 24 h.

Cell Migration Assays
Cell migration assays were performed in the chamber of a 24-well Transwell plate.For migration experiments, starved cells were suspended in a medium without serum with a density of 1 × 10 5 /mL.200 µL of prepared cell suspension; various concentrations of compounds were added to the upper chamber of the chamber and 600 µL of medium (20% FBS) was added to the outer chamber of the chamber.The plates were incubated in an incubator at 37 • C for 48 h.Then, the cells in the upper chamber were swabbed, and the rest of the cells in the chamber were fixed with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet, and photographed (OLYMPUS IX-51) for observation.

Statistical Analysis
All statistical analyses were performed using GraphPad-Prism software 9.All data are expressed as the mean ± SD for three independent tests.The statistical significance of the data between groups was acquired by either Student's test or one-way ANOVA multiple comparisons.

Conclusions
In this study, a new series of SL derivatives (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) were synthesized using epimers 1 and 2 as the parent molecules, isolated from Sphagneticola trilobata, and evaluated for their anti-HCC activity.Furthermore, the structures of 4, 6, and 14 were confirmed by X-ray single-crystal diffraction analyses.The cytotoxic activities of 3-18 on two HCC cell lines, including HepG2 and Huh7, were evaluated using the CCK-8 assay.Among them, compound 10 exhibited the best activity against HepG2 and Huh7 cell lines with IC 50 values of 9.73 and 18.86 µM, respectively.It was suggested that the double bonds at the C-4, 14 or C-3, 4 positions were more favorable to enhancing the activity of HCC.Notably, the double bond between C-11 and C-13 was reduced to the methyl group in all the compounds, which were less potent than the parental compounds, further confirming that SLs' cytotoxic activity is mainly dependent on the α-methylene-γ-lactone group.Further studies showed that 10 induced cell apoptosis, arrested the cell cycle in the S phase, and induced the inhibition of cell proliferation and migration in HepG2 and Huh7.ADME properties prediction showed that 10 may possess the properties to be a drug candidate.Thus, 10 may be a promising lead compound for the treatment of HCC.

Figure 4 .
Figure 4. Compound 10 suppressed the colony formations of Huh7 and HepG2 cells.(A,B) Huh7 and HepG2 cells were treated with 10 for 14 d, and dyed with 0.1% crystal violet.The relative colony numbers are represented as a histogram.

Figure 4 .
Figure 4. Compound 10 suppressed the colony formations of Huh7 and HepG2 cells.(A,B) Huh7 and HepG2 cells were treated with 10 for 14 d, and dyed with 0.1% crystal violet.The relative colony numbers are represented as a histogram.

Figure 5 .
Figure 5. Compound 10 induced apoptosis of Huh7 and HepG2 cells.(A,C) Huh7 and HepG2 cells were treated with 10 for 24 h.The apoptosis was analyzed by flow cytometry.(B,D) The apoptotic rates are represented as a histogram.2.2.4.Compound 10 Arrested the Cell Cycle in the S Phase in Huh7 and HepG2 Cells

Figure 5 . 20 Figure 6 .
Figure 5. Compound 10 induced apoptosis of Huh7 and HepG2 cells.(A,C) Huh7 and HepG2 cells were treated with 10 for 24 h.The apoptosis was analyzed by flow cytometry.(B,D) The apoptotic rates are represented as a histogram.Molecules 2024, 29, x FOR PEER REVIEW 9 of 20

Figure 6 .
Figure 6.Compound 10 induced S phase arrest in Huh7 and HepG2 cells.(A,C) Huh7 and HepG2 cells were incubated with 10 for 24 h, respectively.The distribution of the cell cycle was analyzed by flow cytometry.(B,D) The percentage of cell cycle distribution is represented as a histogram.

Molecules 2024 , 20 Figure 7 .
Figure 7. Experimental results of the migration of cells incubated in compound 10.(A) Wound healing assay results of Huh7 cells incubated in compounds for 0 h and 48 h (scale bar = 100 µm, magnification 4×).(B) Statistical analysis of the scratch experiment and the healing rate (%) = (scratch area at 0 h -scratch area at 24 h)/scratch area at 0 h ×100%.(C) Transwell migration result of Huh7 cells treated with different concentrations of compound 10 for 48 h (scale bar = 100 µm, magnification 20×).(D) Statistical results of Transwell migration tests (n = 3).

Figure 7 .
Figure 7. Experimental results of the migration of cells incubated in compound 10.(A) Wound healing assay results of Huh7 cells incubated in compounds for 0 h and 48 h (scale bar = 100 µm, magnification 4×).(B) Statistical analysis of the scratch experiment and the healing rate (%) = (scratch area at 0 h -scratch area at 24 h)/scratch area at 0 h ×100%.(C) Transwell migration result of Huh7 cells treated with different concentrations of compound 10 for 48 h (scale bar = 100 µm, magnification 20×).(D) Statistical results of Transwell migration tests (n = 3).
IC 50 : concentration of the compound that is required for 50% inhibition of cell growth.